Hepatitis C virus (HCV) is a worldwide health problem, and its treatment and prevention remain a major challenge. Existing therapies are only partially effective and have serious side effects. No vaccine is currently available. RNA interference (RNAi) offers a novel therapeutic approach for treating HCV infections. We have identified a class of shRNAs, called sshRNAs, which have smaller length and appear to act via a somewhat different pathway than ordinary shRNAs or siRNAs. We have further identified very potent sshRNA (IC50 < 10 pM) targeting the HCV internal ribosome entry site (IRES), a highly conserved and essential but undruggable part of the viral RNA. In Phase I, we tested a large number of chemically modified versions of these inhibitors to find types and patterns of modification that would stabilize them in serum without compromising their activity. We identified rules for effective modification and found that such modifications eliminate any stimulation of the innate immune system that can occur with unmodified sshRNAs, particularly if they are blunt-ended. These studies were recently published in two papers in RNA. In Phase II, we will: 1) synthesize chemically modified versions of our alternative lead shRNA using the rules identified in Phase I, and verify their potency and lack of immunostimulatory properties; 2) find optimal combinations of lead shRNAs to minimize shRNA-mediated HCV viral resistant variants; 3) complete screening of nanoparticle formulations to identify the platform that provides the best sshRNA delivery to liver using a mouse reporter model; 4) investigate the toxicity of lead formulated shRNA; and 5) verify the therapeutic efficacy of lead formulated shRNA in an animal model that supports HCV infection. Upon completion of this work, we will be in a very strong position to attract a partner to fund the remaining IND-enabling preclinical studies, file an IND, and proceed with clinical trials. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is one of the most common blood-borne infections and a worldwide health problem. It is a major cause of end-stage liver disease and its treatment and prevention remain a major challenge. RNA interference (RNAi) offers an attractive new approach for treating HCV infections. We have identified chemically modified, highly potent short small hairpin RNAs (sshRNAs) targeting a conserved element of the HCV genome. The goal of this application is the preclinical development of these molecules toward a Phase I clinical trial.